Hepatitis C virus (HCV) is a positive strand RNA virus that establishes both persistent infection in patients and causes cancer. The long-term goal of my research is to elucidate the mechanisms whereby the nonstructural 4B (NS4B) protein facilitates virus production and pathogenesis. There is growing evidence that NS4B facilitates virion production. However, NS4B switch from the replication to the particle formation mode is currently unknown. Further, we know little about the virus and host interactions leading up to NS4B regulation of virion production. Hence, the objective of this application is to gain a better insight into the contribution of NS4B to virus production and the underlying mechanism. We postulate that: [1] During infection, NS4B binds to several virus proteins and regulates their activities, which in the case of NS5A is via p58 formation by host kinases; [2] Specific residues in the NS4B protein contribute to NS5A p58 formation and virion production; [3] JFH1 chimera that are defective in NS5A p58 formation have fewer host kinases interacting with NS5A relative to Wt JFH1. Through a better understanding of NS4B role in virion production, novel antiviral targets for drug development could be uncovered. Thus, we propose the following Specific Aims. Aim 1. Map the NS4B residues required for virus production and NS5A p58 formation; investigate the subcellular distribution of NS5A in the JFH1 chimera; examine the possibility that the adapted viruses have regained the Wt NS5A p58 levels and the underlying mechanism. In this aim, we postulate that specific residues in NS4B are facilitating virus production whereas others are important for HCV replication. Mapping such regions/residues is key to understanding how NS4B can play two important but distinct roles in HCV lifecycle. Hence, we will: [1] Map the NS4B residues required for virus production and NS5A p58 formation, [2] Investigate the subcellular distribution of NS5A p58 during chimeric virus infection and, [3] examine the possibility that the adapted viruses have regained the Wt NS5A p58 levels and the underlying mechanism. Aim 2. Identify virus factors associated NS4B or host factors associated with NS5A p58 and NS4B; examine their roles in virion production. Here, we postulate that NS4B binds to virus factors, leading to a switch from replication to virion production. For instance, NS4B may cause a conformational change in NS5A that favors p58 formation by host kinases. Conversely, NS4B binds to host kinases and causes them to hyperphosphorylate NS5A. Thus, we will: identify [1] NS4B virus/host partners, [2] NS5A p58 host partners and, [3] use surface plasmon resonance to confirm binding and specificity as well as the significance on HCV replication and virion production. We will search for kinases and other host factors associated with NS4B or NSS5A p58. Indeed, kinases regulate various cellular processes and are often targets for treatment of numerous diseases including cancers. Hence, insight into the role of these factors in virion production can lead to the development of novel drugs targeting HCV and the resulting HCC. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is the leading cause of liver transplantation and liver cancer in the United States. Expression of NS4B protein promotes HCV replication complex formation, infectious HCV production and tumor formation in mice. Understanding how NS4B protein interacts with viral and host co-factors to facilitate these events may lead to the development of biomarkers novel antiviral drugs for HCV patients.